This project generates, validates, and delivers novel antibody-drug conjugates (ADCs) that are designed to selectively and potently eradicate chronic lymphocytic leukemia (CLL), the most common leukemia in the U.S., without affecting healthy cells and tissues. CLL is an indolent yet incurable B-cell malignancy that afflicts more than 150,000 men and women and causes more than 4,500 deaths per year in the U.S. alone. There are currently no treatment options for CLL that allow for selective targeting of malignant B cells and that spare healthy B cells and other healthy cells and tissues. With this Premise, the project is built on the Hypothesis that the Fc receptor FCMR, on its own or in combination with other selectively expressed CLL cell surface antigens, can mediate rapid and effective cellular entry of cytotoxic drugs for potent and specific therapeutic intervention. Two independent Specific Aims will be pursued to rigorously test this hypothesis. In Aim 1, a series of molecularly defined ADCs will be generated that deliver and release a highly cytotoxic tubulin inhibitor and a highly cytotoxic DNA-targeting drug, on their own or in combination, via the FCMR internalization and trafficking pathway. These ADCs will be based on the selenomab-drug conjugate platform which utilizes an engineered selenocysteine residue for site-specific drug conjugation. By extensive validation in vitro, ex vivo, and in vivo, a panel of FCMR-targeting selenomab-drug conjugates will be assessed for their stability, specificity, potency, toxicity, and pharmacokinetics. Aim 2 builds on a novel dual variable domain (DVD)-IgG1- based ADC platform that utilizes a unique reactive lysine residue for site-specific drug conjugation. DVD-IgM- based ADCs that can simultaneously engage FCMR and a second CLL cell surface antigen will be built and extensively validated. In addition to a highly modular research strategy that systematically compares different targets, different antibodies, different antibody formats, different linkers, and different drugs, the ex vivo and in vivo experiments in both Specific Aims will be based on peripheral blood mononuclear cells from male and female CLL patients rather than on cell lines to collectively achieve Robust and Unbiased Results toward delivering a candidate for advanced preclinical investigations and eventual clinical translation. Throughout this campaign, conceptually novel biological and chemical components with broad applicability to next-generation ADCs for cancer therapy will be developed.